The present invention relates to novel N-phenyl imidazole derivatives, pharmaceutical compositions containing these compounds and their use as endothelin receptor antagonists.
Endothelin (ET) is a highly potent vasoconstrictor peptide synthesized and released by the vascular endothelium. Endothelin exists as three isoforms, ET-1, ET-2 and ET-3. [Unless otherwise stated xe2x80x9cendothelinxe2x80x9d shall mean any or all of the isoforms of endothelin]. Endothelin has profound effects on the cardiovascular system, and in particular, the coronary, renal and cerebral circulation. Elevated or abnormal release of endothelin is associated with smooth muscle contraction which is involved in the pathogenesis of cardiovascular, cerebrovascular, respiratory and renal pathophysiology. Elevated levels of endothelin have been reported in plasma from patients with essential hypertension, acute myocardial infarction, subarachnoid hemorrhage, atherosclerosis, and patients with uraemia undergoing dialysis.
In vivo, endothelin has pronounced effects on blood pressure and cardiac output. An intravenous bolus injection of ET (0.1 to 3 nmol/kg) in rats causes a transient, dose-related depressor response (lasting 0.5 to 2 minutes) followed by a sustained, dose-dependent rise in arterial blood pressure which can remain elevated for 2 to 3 hours following dosing. Doses above 3 nmol/kg in a rat often prove fatal.
Endothelin appears to produce a preferential effect in the renal vascular bed. It produces a marked, long-lasting decrease in renal blood flow, accompanied by a significant decrease in GFR, urine volume, urinary sodium and potassium excretion. Endothelin produces a sustained antinatriuretic effect, despite significant elevations in atrial natriuretic peptide. Endothelin also stimulates plasma renin activity. These findings suggest that ET is involved in the regulation of renal function and is involved in a variety of renal disorders including acute renal failure, cyclosporine nephrotoxicity, radio contrast induced renal failure and chronic renal failure.
Studies have shown that in vivo, the cerebral vasculature is highly sensitive to both the vasodilator and vasoconstrictor effects of endothelin. Therefore, ET may be an important mediator of cerebral vasospasm, a frequent and often fatal consequence of subarachnoid hemorrhage.
ET also exhibits direct central nervous system effects such as severe apnea and ischemic lesions which suggests that ET may contribute to the development of cerebral infarcts and neuronal death.
ET has also been implicated in myocardial ischemia (Nichols et al. Br. J. Pharm. 99: 597-601, 1989 and Clozel and Clozel, Circ. Res., 65: 1193-1200, 1989) coronary vasospasm (Fukuda et al., Eur. J. Pharm. 165: 301-304, 1989 and Lxc3xcscher, Circ. 83: 701, 1991) heart failure, proliferation of vascular smooth muscle cells, (Takagi, Biochem and Biophys. Res. Commun.; 168: 537-543, 1990, Bobek et al., Am. J. Physiol. 258:408-C415, 1990) and atherosclerosis, (Nakaki et al., Biochem. and Biophys. Res. Commun. 158: 880-881, 1989, and Lerman et al., New Eng. J. of Med. 325: 997-1001, 1991). Increased levels of endothelin have been shown after coronary balloon angioplasty (Kadel et al., No. 2491 Circ. 82: 627, 1990).
Further, endothelin has been found to be a potent constrictor of isolated mammalian airway tissue including human bronchus (Uchida et al., Eur J. of Pharm. 154: 227-228 1988, LaGente, Clin. Exp. Allergy 20: 343-348, 1990; and Springall et al., Lancet, 337: 697-701, 1991). Endothelin may play a role in the pathogenesis of interstitial pulmonary fibrosis and associated pulmonary hypertension, Glard et al., Third International Conference on Endothelin, 1993, p. 34 and ARDS (Adult Respiratory Distress Syndrome), Sanai et al, Supra, p. 112.
Endothelin has been associated with the induction of hemorrhagic and necrotic damage in the gastric mucosa (Whittle et al., Br. J. Pharm. 95: 1011-1013, 1988); Raynaud""s phenomenon, Cinniniello et al., Lancet 337: 114-115, 1991); Crohn""s Disease and ulcerative colitis, Munch et al., Lancet, Vol. 339, p. 381; Migraine (Edmeads, Headache, February 1991 p 127); Sepsis (Weitzberg et al., Circ. Shock 33: 222-227, 1991; Pittet et al., Ann. Surg. 213: 262-264, 1991), Cyclosporin-induced renal failure or hypertension (Eur. J. Pharmacol., 180: 191-192, 1990, Kidney Int, 37: 1487-1491, 1990) and endotoxin shock and other endotoxin induced diseases (Biochem. Biophys. Res. Commun., 161: 1220-1227, 1989, Acta Physiol. Scand. 137: 317-318, 1989) and inflammatory skin diseases. (Clin Res. 41:451 and 484, 1993).
Endothelin has also been implicated in preclampsia of pregnancy. Clark et al., Am. J. Obstet. Gynecol. March 1992, p. 962-968; Kamor et al., N. Eng. J. of Med., Nov. 22, 1990, p. 1486-1487; Dekker et al., Eur J. Ob. and Gyn. and Rep. Bio. 40 (1991) 215-220; Schiffet al., Am. J. Ostet. Gynecol. February 1992, p. 624-628; diabetes mellitus, Takahashi et al., Diabetologia (1990) 33:306-310; and acute vascular rejection following kidney transplant, Watschinger et al., Transplantation Vol. 52, No. 4, pp. 743-746.
Endothelin stimulates both bone resorption and anabolism and may have a role in the coupling of bone remodeling. Tatrai et al. Endocrinology, Vol. 131, p. 603-607.
Endothelin has been reported to stimulate the transport of sperm in the uterine cavity, Casey et al., J. Clin. Endo and Metabolism, Vol. 74, No. 1, p. 223-225, therefore endothelin antagonists may be useful as male contraceptives. Endothelin modulates the ovarian/menstrual cycle, Kenegsberg, J. of Clin. Endo. and Met., Vol. 74, No. 1, p. 12, and may also play a role in the regulation of penile vascular tone in man, Lau et al., Asia Pacific J. of Pharm., 1991, 6:287-292 and Tejada et al., J. Amer. Physio. Soc. 1991, H1078-H1085. Endothelin also mediates a potent contraction of human prostatic smooth muscle, Langenstroer et al., J. Urology, Vol. 149, p. 495-499.
Thus, endothelin receptor antagonists would offer a unique approach toward the pharmacotherapy of hypertension, renal failure, ischemia induced renal failure, sepsis-endotoxin induced renal failure, prophylaxis and/or treatment of radio-contrast induced renal failure, acute and chronic cyclosporin induced renal failure, cerebrovascular disease, myocardial ischemia, angina, heart failure, asthma, pulmonary hypertension, pulmonary hypertension secondary to intrinsic pulmonary disease, atherosclerosis, Raynaud""s phenomenon, ulcers, sepsis, migraine, glaucoma, endotoxin shock, endotoxin induced multiple organ failure or disseminated intravascular coagulation, cyclosporin-induced renal failure and as an adjunct in angioplasty for prevention of restenosis, diabetes, preclampsia of pregnancy, bone remodeling, kidney transplant, male contraceptives, infertility and priaprism and benign prostatic hypertrophy.
This invention comprises compounds represented by Formula (I) and pharmaceutical compositions containing these compounds, and their use as endothelin receptor antagonists which are useful in the treatment of a variety of cardiovascular and renal diseases including but not limited to: hypertension, acute and chronic renal failure, cyclosporine induced nephrotoxicity, benign prostatic hypertrophy, pulmonary hypertension, migraine, stroke, cerebrovascular vasospasm, myocardial ischemia, angina, heart failure, atherosclerosis, and as an adjunct in angioplasty for prevention of restenosis.
This invention further constitutes a method for antagonizing endothelin receptors in an animal, including humans, which comprises administering to an animal in need thereof an effective amount of a compound of Formula (I).
The compounds of this invention are represented by structural Formula (I): 
wherein:
R1 is H, C1-6alkyl or C1-6alkoxy;
R2 is XR5, xe2x80x94R8CO2R4, xe2x80x94(CH2)xC(O)N(R4)S(O)yR9, xe2x80x94(CH2)xS(O)yN(R4)C(O)R9, xe2x80x94CH2)xC(O)N(R4)C(O)R9, xe2x80x94(CH2)nR7, xe2x80x94(CH2)xS(O)yN(R4)S(O)yR9 or Ar;
X is O, S or NR4;
R3 is C1-10alkyl, XC1-10alkyl, Ar or XAr;
R4 is hydrogen or C1-6alkyl;
R5 is xe2x80x94R8CO2R4, xe2x80x94(CH2)xC(O)N(R4)S(O)yR9, xe2x80x94(CH2)xS(O)yN(R4)C(O)R9, xe2x80x94C(O)N(R4)2, xe2x80x94(CH2)xC(O)N(R4)C(O)R9, xe2x80x94(CH2)xS(O)yN(R4)S(O)yR9, xe2x80x94(CH2)nCO2R4, xe2x80x94(CH2)nR7 or Ar;
R6 is C1-8alkyl or xe2x80x94Ar,
P is tetrazol-5-yl, CO2R4 or C(O)N(R4)S(O)yR9;
R7 is C1-6alkoxy, C1-6alkyl, hydroxy, xe2x80x94SC1-6alkyl, xe2x80x94NHSO2R9, SO2NHR9, xe2x80x94SO3H, xe2x80x94CO(NR4)2, CN, xe2x80x94S(O)yC1-6alkyl, xe2x80x94PO(OR4)2, xe2x80x94N(R4)2, xe2x80x94NR4CHO, xe2x80x94NR4COC1-6alkyl, xe2x80x94NR4CON(R4)2 or Ar, or R7 is tetrazolyl, which is substituted or unsubstituted by C1-6alkyl, CF3 or C(O)R3;
R8 is C1-4alkylene, C1-4alkenylene or C1-4alkylidene, all of which may be linear or branched;
R9 is C1-10alkyl, N(C1-8alkyl)2 or Ar;
n is 1 to 4;
m is 0 to 3;
x is 0 to 4;
y is 1 or 2;
Ar is: 
xe2x80x83naphthyl, indolyl, pyridyl, thienyl, oxazolidinyl, thiazolyl, isothiazolyl, pyrazolyl, imidazolyl, imidazolinyl, thiazolidinyl, isoxazolyl, oxadiazolyl, thiadiazolyl, morpholinyl, piperidinyl, piperazinyl, pyrrolyl, or pyriridyl; all of which may be substituted or unsubstituted by one or more Z1 or Z2 groups;
Z1 and Z2 are independently hydrogen, XR4, C1-8alkyl, CO2R4, C(O)N(R4)2, CN, NO2, F, Cl, Br, I, N(R4)2, NHC(O)R4 or tetrazolyl which may be substituted or unsubstituted by C1-6alkyl, CF3 or C(O)R4;
A is Cxe2x95x90O or [C(R4)2]q;
B is xe2x80x94CH2xe2x80x94 or xe2x80x94Oxe2x80x94; and
q is 1 or 2;
and the dotted line indicates the optional presence of a double bond;
or a pharmaceutically acceptable salt thereof;
provided:
R6 is not thienyl; and
R2 is not (CH)nC1-6alkyl, or xe2x80x94CHxe2x95x90CHCO2R4.
All alkyl, alkenyl, alkynyl and alkoxy groups may be straight or branched.
The compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active form. All of these compounds and diastereoisomers are contemplated to be within the scope of the present invention.
Preferred compounds are those wherein there is an optional double bond present; R4 is cis to P; R1 is H or C1-6alkoxy; R2 is xe2x80x94OR5, xe2x80x94R8CO2H, xe2x80x94(CH2)xC(O)N(R4)S(O)yR9, xe2x80x94(CH2)xC(O)NHC(O)R9, xe2x80x94(CH2)nR7, or R2 is phenyl or pyridyl, both of which may be substituted or unsubstituted by one or more Z1 or Z2 groups; R3 is C1-10alkyl, C1-10alkoxy or R3 is phenyl or pyrazolyl, both of which may be substituted or unsubstituted by C1-6 alkoxy, Cl, Br, F or I; R4 is hydrogen or C1-6alkyl; R5 is xe2x80x94R8CO2H, xe2x80x94(CH2)xC(O)N(R4)S(O)yR 9, xe2x80x94(CH2)xC(O)NHC(O)R9, xe2x80x94(CH2)nCO2R4, xe2x80x94(CH2)nR7 or pyridyl which may be substituted or unsubstituted by Z1; R6 is (a), (b) or indolyl; P is CO2H or C(O)NHS(O)yR9; R7 is C1-6alkoxy, C1-6alkyl, piperidinyl, hydroxy, xe2x80x94NHSO2R9, xe2x80x94CONHR4, xe2x80x94N(R4)2, xe2x80x94NR4CON(R4)2 or R7 is thienyl, pyridyl, pyrimidyl, phenyl, all of which may be substituted or unsubstituted by one or more Z1 or Z2 groups or R7 is tetrazol-5-yl, piperazinyl both of which are substituted or unsubstituted by C1-6alkyl; R8 is C1-4alkenylene which may be linear or branched; R9 is C1-10alkyl, N(C1-8alkyl)2 or phenyl which may be substituted or unsubstituted by C1-8alkyl; n is 1 to 4; m is 1; x is 0 to 4; y is 1 or 2; Z1 and Z2 are independently hydrogen, hydroxy, C1-8alkyl, C1-6alkoxy, CO2H, C(O)N(R4)2, F, Cl, Br, I, N(R4)2, or tetrazolyl which may be substituted or unsubstituted by C1-6alkyl; A is [C(R4)2]q; q is 1; and B is xe2x80x94Oxe2x80x94.
More preferred are compounds wherein there is an optional double bond present; R4 is cis to P; R1 is H or C1-3alkoxy; R2 is xe2x80x94(CH2)xC(O)NHS(O)2R9, xe2x80x94OR5, xe2x80x94(CH2)nR7, or xe2x80x94O(CH2)1-3CO2H; R3 is C1-5alkyl; R4 is hydrogen; R5 is xe2x80x94(CH2)xC(O)NHS(O)2R9, xe2x80x94(CH2)xC(O)NHC(O)R9, xe2x80x94(CH2)nR7 or pyridyl which may be substituted or unsubstituted by Z1; R6 is (b); P is CO2H; R7 is hydroxy, xe2x80x94NHSO2R9, xe2x80x94CONH(C1-5alkyl), or R7 is tetrazol-5-yl or piperazinyl, both of which may be substituted or unsubstituted by C1-6alkyl or R7 is thienyl, pyridyl, pyrimidyl, or phenyl, all of which may be substituted or unsubstituted by one or more Z1 or Z2 groups; R9 is C1-5alkyl, N(C1-5alyl)2 or R9 is phenyl which may be substituted or unsubstituted by C1-5alkyl; n is 1 to 4; m is 1; x is 0 to 4; Z1 and Z2 are independently hydrogen, hydroxy, C1-6alkoxy, CO2H, C(O)NH2, F, Cl, or tetrazolyl which may be substituted or unsubstituted by C1-6alkyl; A is xe2x80x94CH2xe2x80x94; and B is xe2x80x94Oxe2x80x94.
Especially preferred are the following compounds:
(E)-3-[2-Butyl-1-[2-[N-(phenylsulfonyl)]carboxamidomethoxy-4-methoxyphenyl]-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxy)phenylmethyl]-2-propenoic acid dipotassium;
(E)-3-[2-Butyl-1-[2-(tetrazol-5-yl)methoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
(E)-3-[2-Butyl-1-[2-(2-carboxyphenyl)methoxy-4-methoxy]phenyl-1H-imdazol-5-yl]-2-[(2-methoxy-4,5-metlhylenedioxyphenyl)methyl]-2-propenoic acid;
(E)-3-[2-Butyl-1-[2-(4-carboxyphenyl)methoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(3-carboxyphenyl)methoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-(2-Butyl-1-[2-[N-(2-methylphenyl)sulfonyl]carboxamidomethoxy-4-methoxy]phenyl-1H-imidazol-5-yl-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-[N-(4-methylphenyl)sulfonyl]carboxamidomethoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(N-dimethylaminosulfonyl)carboxamidomethoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(N-methanesulfonyl)carboxamidomethoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(N-t-butylsulfonyl)carboxamidomethoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(N-i-propylsulfonyl)carboxamidomethoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(2-(tetrazol-5-yl)benzyloxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(2-ethyl-3H-tetrazol-5-yl)benzyloxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-[(4-carboxypyridin-3-yl)oxy]4-methoxy]phenyl-1H-imidazol-5yl]-2-[(3,4-methylenedioxyphenyl)methyl]-2-propenoic acid;
(E)-3-[2-Butyl-[1-(2-carboxymethoxy)-4-methoxy)phenyl]-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
E-3-[2-Butyl-1-[2-(3-carboxy)propoxy-4-methoxy]phenyl-1H-imidazol-5-yl]-2-[(2-methoxy-4,5-methylenedioxyphenyl)methyl]-2-propenoic acid;
The present invention provides compounds of Formula (I), 
which can be prepared by reacting an aniline of Formula (2) 
with an iminoether of Formula (3) 
in a solvent such as dichloromethane at reflux to afford an amidine of Formula (4). 
An iminoether of Formula (3) may be prepared from a nitrile of Formula (5)
R3xe2x80x94CNxe2x80x83xe2x80x83(5)
by reaction with methanolic hydrogen chloride, in a solvent such as methyl alcohol, to provide the corresponding iminoether hydrochloride followed by liberation of (3) by treatment with a base such as triethylamine in a solvent such as diethyl ether. Filtration of the resulting.product to remove triethylamine hydrochloride followed by evaporation of the solvent in vacuo provides (3).
Reaction of an amidine of Formula (4) with 2-bromo-malondialdehyde (6) 
in a solvent such as isopropanol containing triethylammonium acetate at reflux affords an aldehyde of Formula (7). 
Knoevenagel condensation of a compound such as (7) with a half acid of Formula (8), wherein R6 is Ar, m is 1 or 2, and R10 is C1-8alkyl, 
in a solvent such as benzene at reflux, in the presence of piperidinium acetate with azeotropic removal of water using a Dean-Stark apparatus, affords an ester of Formula (9). 
Saponification of an ester of Formula of (9) with aqueose sodium hydroxide in a solvent such as ethanol following acidic work up affords a compound of Formula of (1), where R4 is H, and P is COOH.
Alternatively, hydrogenation of a compound of Formula (9) with hydrogen gas under pressure at approximately 60 psi in the presence of a suitable catalyst such as 10% palladium on charcoal in a suitable solvent such as ethyl acetate or ethanol affords a compound of Formula (10); 
Saponification of an ester of Formula of (10) with aqeouse sodium hydroxide in a solvent such as ethanol following acidic work up affords a compound of Formula of (1), where there is an optional single bond, R4 is H, P is COOH.
An aniline of Formula (11) may be prepared by reaction of a nitrophenol of Formula (13) 
with bromomethyl methyl ether in a solvent such as dimethyl formamide in the presence of a base such as sodium hydride to afford an ether of Formula (14). 
Reduction of an ether of Formula (14) using hydrogen in the presence of a catalyst such as 10% palladium on charcoal in a solvent such as ethanol affords a aniline of Formula (11). 
For compounds of Formula (I), wherein R2 is O(CH2)nR7 (R7 is tetrazole or Ar) or OCH2CONHSO2R6, R4 is hydrogen, P is CO2H, an aniline of Formula (11 is reacted as described above to provide an aldehyde of Formula (12). 
A malonic acid half ester of Formula (8), where R6 is (b), A is xe2x80x94CH2xe2x80x94, B is xe2x80x94Oxe2x80x94, and m is 1, may be prepared from an aldehyde such as (15) 
by treatment with a dialkyl malonate of Formula (16), wherein R10 is C1-8alkyl,
CH2(CO2R10)2xe2x80x83xe2x80x83(16)
in a solvent such as cyclohexane at reflux, in the presence of a base such as piperidine containing a catalytic amount of para-toluic acid, with removal of water using a Dean-Stark apparatus to afford a product of Formula (17). 
Reduction of a compound of Formula (17) with sodium borohydride in a solvent such as ethanol affords a product of Formula (18). 
Mono saponification of an ester of Formula (18) with aqueous potassium hydroxide in a solvent such as ethanol followed by acidification with aqueous hydrochloric acid affords a malonic acid derivative of Formula (8), where R6 is (b), A is xe2x80x94CH2xe2x80x94, B is xe2x80x94Oxe2x80x94, m is 1.
Reaction of an aldehyde of Formula (12) with a half acid of Formula (8) by the Knoevenagel procedure described above affords an acrylate of Formula (19). 
Deprotection of an ether of Formula (19) using ethanolic hydrogen chloride affords a phenol of Formula (20). 
Alkylation of a phenol of Formula (20) using an allyl chloroacetate of Formula (21)
ClCH2CO2Allylxe2x80x83xe2x80x83(21)
in a solvent such as dimethylformamide using a base such as anhydrous potassium carbonate provides a mixed ester of Formula (22). 
Selective cleavage of an allyl ester of Formula (22) using triethylsilane in the presence of a catalyst such as triphenylphosphine palladium(0) in a solvent such as dichloromethane provides a mono acid of Formula (23). 
Coupling a mono acid of formula (23) with a sulfonamide of Formula (24)
R9SO2NH2xe2x80x83xe2x80x83(24)
in the presence of a catalyst such as 4-dimethylaminopyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride in a solvent such as dichloromethane at reflux affords a sulfonamide of Formula of (25). 
Saponification of an ester of Formula (25) using aqueous sodium hydroxide in a solvent such as ethanol affords, after acidification with aqueous hydrochloric acid, an acid of Formula (I), wherein R2 is OCH2CO2NHSO2R9, A is xe2x80x94CH2xe2x80x94, B is xe2x80x94Oxe2x80x94, R4 is hydrogen, and P is CO2H.
Alternatively, a phenol of Formula (20) may by alkylated with chloroacetonitrile in a solvent such as dimethylformamide to produce a compound of Formula (26). 
Reaction of a nitrile of Formula (26) with sodium azide in the presence of trimethyltin chloride in a solvent such as toluene at elevated temperature affords a tetrazole of Formula (27). 
Saponification of an ester of Formula (27) using aqueous sodium hydroxide in a solvent such as ethanol affords, after acidification with aqueous hydrochloric acid, an acid of Formula (I), wherein R2 is OCH2(tetrazol-5-yl), A is xe2x80x94CH2xe2x80x94, B is xe2x80x94Oxe2x80x94, R4 is hydrogen, and P is CO2H.
Alternatively, a phenol of Formula (20) may also be alkylated with an alkyl halide of Formula (28), where X is I, Br, or Cl;
R7(CH2)nXxe2x80x83xe2x80x83(28)
to provide an ether of Formula (29). 
Saponification of an ester of Formula (29) using aqueous sodium hydroxide in a solvent such as ethanol affords, after acidification with aqueous hydrochloric acid, an acid of Formula (I), wherein R2 is O(CH2)nR7, A is xe2x80x94CH2xe2x80x94, B is xe2x80x94Oxe2x80x94, R4 is hydrogen, P is CO2H.
In order to use a compound of the Formula (I) or a pharmaceutically acceptable salt thereof for the treatment of humans and other mammals it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
Compounds of Formula (I) and their pharmaceutically acceptable salts may be administered in a standard manner for the treatment of the indicated diseases, for example orally, parenterally, sub-lingually, transdermally, rectally, via inhalation or via buccal administration.
Compounds of Formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as syrups, tablets, capsules and lozenges. A syrup formulation will generally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, peanut oil, olive oil, glycerine or water with a flavouring or colouring agent. Where the composition is in the form of a tablet, any pharmaceutical carrier routinely used for preparing solid formulations may be used. Examples of such carriers include magnesium stearate, terra alba, talc, gelatin, agar, pectin, acacia, stearic acid, starch, lactose and sucrose. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the form of a soft gelatin shell capsule any pharmaceutical carrier routinely used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils and are incorporated in a soft gelatin capsule shell.
Typical parenteral compositions consist of a solution or suspension of the compound or salt in a sterile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the form of an aerosol using a conventional propellant such as dichlorodifluoromethane or trichlorofluoromethane.
A typical suppository formulation comprises a compound of Formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent, for example polymeric glycols, gelatins, cocoa-butter or other low melting vegetable waxes or fats or their synthetic analogues.
Typical transdermal formulations comprise a conventional aqueous or non-aqueous vehicle, for example a cream, ointment, lotion or paste or are in the form of a medicated plaster, patch or membrane.
Preferably the composition is in unit dosage form, for example a tablet, capsule or metered aerosol dose, so that the patient may administer to themselves a single dose.
Each dosage unit for oral administration contains suitably from 0.1 mg to 500 mg/Kg, and preferably from 1 mg to 100 mg/Kg, and each dosage unit for parenteral administration contains suitably from 0.1 mg to 100 mg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. Each dosage unit for intranasal administration contains suitably 1-400 mg and preferably 10 to 200 mg per person. A topical formulation contains suitably 0.01 to 1.0% of a compound of Formula (I).
The daily dosage regimen for oral administration is suitably about 0.01 mg/Kg to 40 mg/Kg, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. The daily dosage regimen for parenteral administration is suitably about 0.001 mg/Kg to 40 mg/Kg, of a compound of the Formula (I) or a pharmaceutically acceptable salt thereof calculated as the free acid. The daily dosage regimen for intranasal administration and oral inhalation is suitably about 10 to about 500 mg/person. The active ingredient may be administered from 1 to 6 times a day, sufficient to exhibit the desired activity.
No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.
The biological activity of the compounds of Formula (I) are demonstrated by the following tests:
A) Membrane Preparation
Rat cerebellum or kidney cortex were rapidly dissected and frozen immediately in liquid nitrogen or used fresh. The tissues, 1-2 g for cerebellum or 3-5 g for kidney cortex, were homogenized in 15 mls of buffer containing 20 mM Tris HCl and 5 mM EDTA, pH 7.5 at 4xc2x0 C. using a motordriven homogenizer. The homogenates were filtered through cheesecloth and centrifuged at 20,000xc3x97g for 10 minutes at 4xc2x0 C. The supernatant was removed and centrifuged at 40,000xc3x97g for 30 minutes at 4xc2x0 C. The resulting pellet was resuspended in a small volume of buffer containing 50 mM Tris, 10 mM MgCl2, pH 7.5; aliquotted with small vials and frozen in liquid nitrogen. The membranes were diluted to give 1 and 5 mg of protein for each tube for cerebellum and kidney cortex in the binding assay.
Freshly isolated rat mesenteric artery and collateral vascular bed were washed in ice cold saline (on ice) and lymph nodes were removed from along the major vessel. Then, the tissue was homogenized using a polytron in buffer containing 20 mM Tris and 5 mM EDTA, pH 7.5 at 4xc2x0 C. in 15 ml volume for xcx9c6 gm of mesenteric artery bed. The homogenate was strained through cheesecloth and centrifuged at 2,000xc3x97g for 10 min. at 4xc2x0 C. The supernatant was removed and centrifuged at 40,000xc3x97g for 30 min. at 4xc2x0 C. The resulting pellet was resuspended as explained above for cerebellum and kidney cortex. Approximately 10 mg of membrane protein was used for each tube in binding experiments.
B) [125I]ET-1 Binding Protocol
[125I]ET-1 binding to membranes from rat cerebellum (2-5 mg protein/assay tube) or kidney cortex (3-8 mg protein/assay tube) were measured after 60 minutes incubation at 30xc2x0 C. in 50 mM Tris HCl, 10 mM MgCl2, 0.05% BSA, pH 7.5 buffer in a total volume of 100 ml. Membrane protein was added to tubes containing either buffer or indicated concentration of compounds. [125I]ET-1 (2200 Ci/mmol) was diluted in the same buffer containing BSA to give a final concentration of 0.2-0.5 nM ET-1. Total and nonspecific binding were measured in the absence and presence of 100 nM unlabelled ET-1. After the incubation, the reactions were stopped with 3.0 ml cold buffer containing 50 mM Tris and 10 mM MgCl2, pH 7.5. Membrane bound radioactivity was separated from free ligand by filtering through Whatman GF/C filter paper and washing the filters 5 times with 3 ml of cold buffer using a Brandel cell harvester. Filter papers were counted in a gamma counter with an efficiency of 75%.
Rat aorta are cleaned of connective tissue and adherent fat, and cut into ring segments approximately 3 to 4 mm in length. Vascular rings are suspended in organ bath chambers (10 ml) containing Krebs-bicarbonate solution of the following composition (millimolar): NaCl, 112.0; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; and dextrose, 11.0. Tissue bath solutions are maintained at 37xc2x0 C. and aerated continuously with 95% O2/5% CO2. Resting tensions of aorta are maintained at 1 g and allowed to equilibrate for 2 hrs., during which time the bathing solution is changed every 15 to 20 min. Isometric tensions are recorded on Beckman R-611 dynographs with Grass FT03 force-displacement transducer. Cumulative concentration-response curves to ET-1 or other contractile agonists are constructed by the method of step-wise addition of the agonist ET-1 concentrations are increased only after the previous concentration produces a steady-state contractile response. Only one concentration-response curve to ET-1 is generated in each tissue. ET receptor antagonists are added to paired tissues 30 min prior to the initiation of the concentration-response to contractile agonists.
ET-1 induced vascular contractions are expressed as a percentage of the response elicited by 60 mM KCl for each individual tissue which is determined at the beginning of each experiment. Data are expressed as the meanxc2x1S.E.M. Dissociation constants (Kb) of competitive antagonists were determined by the standard method of Arunlakshana and Schild.